Gene expression profile and synovial microcirculation at early stages of collagen-induced arthritis

Philip Gierer 1 2 Saleh Ibrahim 0 Thomas Mittlmeier 2 Dirk Koczan 0 Steffen Moeller 0 Jrgen Landes 3 Georg Gradl 1 2 Brigitte Vollmar 2 0 Institute of Immunology, University of Rostock , Rostock , Germany 1 Department of Trauma & Reconstructive Surgery, University of Rostock , Rostock , Germany 2 Department of Experimental Surgery, University of Rostock , Rostock , Germany 3 Department of Surgery, Klinikum Innenstadt, Ludwig Maximilians University , Munich , Germany A better understanding of the initial mechanisms that lead to arthritic disease could facilitate development of improved therapeutic strategies. We characterized the synovial microcirculation of knee joints in susceptible mouse strains undergoing intradermal immunization with bovine collagen II in complete Freund's adjuvant to induce arthritis (i.e. collageninduced arthritis [CIA]). Susceptible DBA1/J and collagen II Tcell receptor transgenic mice were compared with CIA-resistant FVB/NJ mice. Before onset of clinical symptoms of arthritis, in vivo fluorescence microscopy of knee joints revealed marked leucocyte activation and interaction with the endothelial lining of synovial microvessels. This initial inflammatory cell response correlated with the gene expression profile at this disease stage. - Introduction Murine collagen-induced arthritis (CIA) is a chronic inflammatory disease that bears all the hallmarks of rheumatoid arthritis (RA), namely polyarthritis and synovitis with subsequent cartilage and bone erosions [1]. CIA is induced in susceptible strains of mice (e.g. DBA1/J) by immunization with bovine collagen type II in complete Freund's adjuvant (CFA). The development of CIA is thought to depend on T cells, and disease susceptibility is linked to the major histocompatibility region [2]. Activated lymphocytes migrate to the joint, where an inflammatory cascade involving T cells, macrophages, monocytes, B cells and activated synoviocytes is triggered. This cellular infiltration, together with production of a complex array of cytokines and other soluble mediators, contributes to synovial proliferation, pannus formation, cartilage destruction and subchondral bone erosion [3]. Because the inflammatory process within joint tissues represents a key feature of RA, an understanding of the mechanisms that induce and sustain this aspect of RA pathology would permit development of new and powerful therapeutic strategies. With direct online visualization, the technique of intravital fluorescence microscopy permits dissection of the complex cell inflammatory response, with differentiation between cellular subtypes and their distinct adhesion molecule dependent interactions within the microcirculation. AIA = antigen-induced arthritis; CCL = CC chemokine ligand; CFA = complete Freund's adjuvant; CIA = collagen-induced arthritis; GO = Gene Ontology; IL = interleukin; mBSA = methylated bovine serum albumin; NCF = neutrophil cytosolic factor; RA = rheumatoid arthritis; TCR = T-cell receptor; TNF = tumour necrosis factor; VRBC = centre line red blood cell velocity. The approach of in vivo microscopy has successfully been applied in joints of mice with antigen-induced arthritis (AIA) [4]. AIA is established in mice by immunizing them with methylated bovine serum albumin (mBSA) in CFA with or without an arthritogenic infectious agent at days 0 and 7, followed by intra-articular injection of mBSA at day 21 [5]. Although AIA is an established animal model for the study of human RA [6], arthritis is more commonly induced using collagen, and this represents the primary animal model for RA in humans [7-9]. Therefore, we employed in vivo microcirculatory analysis of knee joints in mice with CIA, using different strains that are known to acquire CIA, such as DBA1/J mice and T-cell receptor (TCR) transgenic mice that carry the rearranged V11.1 and V8.2 chain genes isolated from a type II collagen-specific T-cell hybridoma (DBA-CII-TCR Tg) [10]. FVB/NJ mice were used as controls, because these mice have been reported to be resistant to arthritis induction, probably because of a genomic deletion of TCR V gene segments [11]. Because we were particularly interested in the disease initiation stage, the synovial microcirculation was assessed before the onset of clinical symptoms of arthritis. We further characterized the global gene expression profile at this early stage in the disease in order to define the initial molecular mechanisms and to determine the onset of joint inflammation. Materials and methods Animal model The experimental protocol was approved by the local animal rights protection authorities (LVL M-V/TSD/7221.3-1.1-037/ 04) and followed the National Institutes of Health guidelines for the care and use of laboratory animals. DBA1/J and FVB/ NJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Collagen II specific TCR transgenic mice were a kind gift from Professor Ladiges, University of Washington, USA [10]. All mice were kept under standard conditions at the animal care facility of the University of Rostock. Mice aged 8 weeks (n = 510 per strain and group) were immunized intradermally at the base of the tail with 125 g bovine collagen II (Chondrex, Redmond, WA, USA) emulsified in CFA (DIFCO, Detroit, MI, USA) or with equivalent volumes of CFA only. Six weeks after immunization and before clinical signs of arthritis manifested, animals were anaesthetized with ketamine (90 mg/kg body weight) and xylacin (6 mg/kg) and placed on a heating pad to maintain their body temperature at 37C. A catheter was placed in the left jugular vein for application of fluorescent dyes. For in vivo multifluorescence microscopy of synovial microcirculation, we used the knee joint model initially described by Veihelmann and coworkers [4]. Briefly, the skin was incised distal to the patella tendon. After removal of the overlying soft tissues, the patella tendon was transversally cut and the proximal and distal part carefully mobilized. After exposure, the 'Hoffa's fatty body' was superfused with 37C warm physiological saline solution to prevent the tissues from drying and finally covered with a glass slide. Following a 15-min stabilization period after surgical preparation, in vivo microscopy of the synovial tissue was performed. At the end of the experiments, animals were killed by exsanguination. The complete knee joint was excised and harvested for subsequent histology. Paws were used for gene expression profile analysis. Clinical evaluation of arthritis As described by Nanakumar and coworkers [12], scoring of animals was done blindly using a scoring system based on the number of inflamed joints in each paw, inflammation being defined by swelling and redness. In this scoring system each inflamed toe or knuckle is attributed 1 point, whereas an inflamed wrist or ankle is attributed 5 points, resulting in a score of 0 to 15 (five toes + five knuckles + one wrist/ankle) for each paw and 060 points for each mou (...truncated)